U.S. patent number 9,101,564 [Application Number 13/261,236] was granted by the patent office on 2015-08-11 for heparanase activity inhibitor, wrinkle improving agent containing same, and pharmaceutical composition.
This patent grant is currently assigned to Shiseido Company, Ltd.. The grantee listed for this patent is Satoshi Amano, Hirotada Fukunishi, Shunsuke Iriyama, Masaru Suetsugu. Invention is credited to Satoshi Amano, Hirotada Fukunishi, Shunsuke Iriyama, Masaru Suetsugu.
United States Patent |
9,101,564 |
Iriyama , et al. |
August 11, 2015 |
Heparanase activity inhibitor, wrinkle improving agent containing
same, and pharmaceutical composition
Abstract
A heparanase activity inhibitor containing, as an active
ingredient, a 4-alkylresorcinol represented by formula (I):
##STR00001## wherein R represents a C1-6 linear or branched alkyl
group.
Inventors: |
Iriyama; Shunsuke (Yokohama,
JP), Fukunishi; Hirotada (Yokohama, JP),
Suetsugu; Masaru (Yokohama, JP), Amano; Satoshi
(Yokohama, JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
Iriyama; Shunsuke
Fukunishi; Hirotada
Suetsugu; Masaru
Amano; Satoshi |
Yokohama
Yokohama
Yokohama
Yokohama |
N/A
N/A
N/A
N/A |
JP
JP
JP
JP |
|
|
Assignee: |
Shiseido Company, Ltd. (Tokyo,
JP)
|
Family
ID: |
43826307 |
Appl.
No.: |
13/261,236 |
Filed: |
September 29, 2010 |
PCT
Filed: |
September 29, 2010 |
PCT No.: |
PCT/JP2010/066997 |
371(c)(1),(2),(4) Date: |
March 27, 2012 |
PCT
Pub. No.: |
WO2011/040495 |
PCT
Pub. Date: |
April 07, 2011 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20120184782 A1 |
Jul 19, 2012 |
|
Foreign Application Priority Data
|
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|
|
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Sep 30, 2009 [JP] |
|
|
2009-228209 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
35/04 (20180101); A61Q 19/08 (20130101); A61K
31/05 (20130101); A61P 17/02 (20180101); A61P
43/00 (20180101); A61P 9/00 (20180101); A61P
35/00 (20180101); A61P 17/00 (20180101); A61K
8/347 (20130101); A61K 2800/782 (20130101) |
Current International
Class: |
A61K
8/00 (20060101); A61K 31/05 (20060101); A61K
8/34 (20060101); A61Q 19/08 (20060101) |
Field of
Search: |
;424/78.03 ;568/766
;514/18.6,18.7,18.8 |
References Cited
[Referenced By]
U.S. Patent Documents
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4959393 |
September 1990 |
Torihara et al. |
5614178 |
March 1997 |
Bloom et al. |
|
Foreign Patent Documents
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0 341 664 |
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Nov 1989 |
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EP |
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02-049715 |
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Feb 1990 |
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JP |
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02-292213 |
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Dec 1990 |
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JP |
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07-206669 |
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Aug 1995 |
|
JP |
|
2001-302505 |
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Oct 2001 |
|
JP |
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2006-016343 |
|
Jan 2006 |
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JP |
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2006-124358 |
|
May 2006 |
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JP |
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2007-509966 |
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Apr 2007 |
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JP |
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2007-254412 |
|
Oct 2007 |
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JP |
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WO 2005/042712 |
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May 2005 |
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WO |
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WO 2009/122540 |
|
Oct 2009 |
|
WO |
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WO 2009/123215 |
|
Oct 2009 |
|
WO |
|
Other References
Ilan et al., "Regulation, function and clinical significance of
heparanase in cancer metastatis and angiogenesis," The
International Journal of Biochemistry & Cell Biology, 2006,
38:2018-2039. cited by applicant .
Vlodavsky et al., "Mammalian heparanase: involvement in cancer
metastasis, angiogenesis and normal development," Cancer Biology,
2002, 12:121-129. cited by applicant .
Zcharia et al., "Heparanase accelerates wound angiogenesis and
wound healing in mouse and rat models," The FASEB Journal, Feb.
2005, 19:211-221. cited by applicant .
Lipkin, E. Alfred, "Hexylresorcinol in the Treatment of Arthritis,"
The Lancet, Letters to the Editor, Aug. 24, 1957, 385-386. cited by
applicant.
|
Primary Examiner: Katakam; Sudhakar
Assistant Examiner: Bakshi; Pancham
Attorney, Agent or Firm: Foley & Lardner LLP
Claims
The invention claimed is:
1. A method for suppressing formation of wrinkles caused by a
reduced level of heparan sulfate in an epidermal basal membrane,
comprising administering, to a subject in need thereof, an
effective amount of 4-isobutylresorcinol as an active
ingredient.
2. A method for inhibiting heparanase activity, comprising
administering, to a subject in need thereof, 4-isobutylresorcinol
as an active ingredient.
3. The method according to claim 1, wherein heparanase activity is
inhibited by the administration of the 4-isobutylresorcinol.
4. The method according to claim 1, wherein the
4-isobutylresorcinol is administered by oral administration,
parenteral administration or local administration.
5. The method according to claim 3, wherein the
4-isobutylresorcinol is administered by oral administration,
parenteral administration or local administration.
6. The method according to claim 4, wherein 4-isobutylresorcinol is
present in an external preparation for skin in the range of at
least 0.0001 to 1 mass %, as the dry mass of the total external
preparation.
7. The method according to claim 5, wherein 4-isobutylresorcinol is
present in an external preparation for skin in the range of at
least 0.0001 to 1 mass %, as the dry mass of the total external
preparation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage application of
PCT/JP2010/066997, filed Sep. 29, 2010, which claims priority from
Japanese application JP 2009-228209, filed Sep. 30, 2009.
TECHNICAL FIELD
The present invention relates to a heparanase activity inhibitor
containing a 4-alkylresorcinol as an active ingredient, and to a
wrinkle improving agent and a pharmaceutical composition employing
the heparanase activity inhibitor.
BACKGROUND ART
Heparanase is present in a variety of cells such as platelets,
leukocytes, endothelial cells and smooth muscle cells, as an enzyme
that specifically degrades heparan sulfate chains in various types
of heparan sulfate proteoglycan. In the skin, in particular, it is
produced by epidermal keratinocytes composing the epidermis and
fibroblasts or vascular endothelial cells of the dermis. Its
production is also known to be elevated in various types of cancer
cells.
Heparan sulfate proteoglycan (HSPG), which is degraded by
heparanase, is a polymer found in various animal tissue cell
surfaces and extracellular matrices, and it is known to have
functions including extracellular buildup of heparan
sulfate-binding growth factors (bFGF (basic fibroblast growth
factor), HGF (hepatocyte growth factor), VEGF (vascular endothelial
growth factor), HB-EGF (heparin binding EGF-like growth factor),
and the like).
Perlecan, a type of heparan sulfate proteoglycan, is also present
in the epidermal basal membrane at the interface between the
epidermis and dermis, and it binds heparan sulfate-binding growth
factors to the epidermal basal membrane, controlling migration of
growth factors between the epidermis and dermis. Perlecan also
controls growth factors for epidermal basal cells that bind to the
basal membrane, and it has been shown to be essential for proper
growth and differentiation of the epidermis. Consequently,
decomposition of perlecan heparan sulfate chains by activation or
accelerated expression of heparanase disturbs release of
accumulated growth factors and control of growth factors in the
epidermis and dermis, leading to failure of control of
differentiation and growth of the epidermis and thickening of the
dermis, and promoting formation of wrinkles (see
PCT/JP2009/056717). In other words, inhibition of heparanase
activity suppresses the release of growth factors that accompanies
decomposition of heparan sulfate, and allows migration of growth
factors between the epidermis and dermis to be controlled, thereby
aiding in anti-aging of the skin.
A link between heparanase and cancer malignancy has also been
suggested. In particular, it is known that cancer cells with
increased production of heparanase have higher proliferative and
metastatic ability, and increased inducibility of angiogenesis
(Non-patent document 1). Heparanase is also known to have a
function of accelerating wound healing (Non-patent document 2).
Therefore, effective inhibition of heparanase activity is effective
for purposes including suppressing proliferation or metastasis of
cancer cells, and suppressing angiogenesis.
PRIOR ART DOCUMENTS
Non-Patent Documents
Non-patent document 1: Vlodaysky I., et al., Semin Cancer Biol.,
2002; 12(2):121-129 Non-patent document 2: Zacharia E., et al.,
FASEB J. 2005 February; 19(2):211-21
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
In light of the background explained above, it has been a goal to
find substances that can effectively inhibit heparanase
activity.
It is an object of the present invention, which has been
accomplished in consideration of the above, to provide a heparanase
activity inhibitor that can effectively inhibit heparanase
activity, and a wrinkle improving agent and a pharmaceutical
composition employing the heparanase activity inhibitor.
Means for Solving the Problems
As a result of much diligent research, the present inventors found
that certain 4-alkylresorcinols effectively inhibit heparanase
activity.
Specifically, the gist of the present invention is as follows. (1)
A heparanase activity inhibitor containing, as an active
ingredient, a 4-alkylresorcinol represented by formula (I):
##STR00002## wherein R represents a C1-6 linear or branched alkyl
group. (2) A heparanase activity inhibitor according to (1),
wherein R in formula (I) is a C2-4 linear or branched alkyl group.
(3) A heparanase activity inhibitor according to (2), wherein the
4-alkylresorcinol of formula (I) is 4-isobutylresorcinol. (4) A
pharmaceutical composition for treatment, improvement or prevention
of a condition or symptom associated with heparanase activity, the
pharmaceutical composition containing, as an active ingredient, a
heparanase activity inhibitor according to any one of (1) to (3).
(5) A pharmaceutical composition according to (4), which is used
for wound healing, suppression of proliferation or metastasis of
cancer cells, or suppression of angiogenesis. (6) A wrinkle
improving agent that prevents or suppresses formation of wrinkles,
the wrinkle improving agent containing, as an active ingredient, a
heparanase activity inhibitor according to any one of (1) to
(3).
Effect of the Invention
Since the heparanase activity inhibitor of the invention can
efficiently inhibit heparanase activity, it can be suitably used,
for example, as an active ingredient in a wrinkle improving agent,
to prevent or suppress formation of wrinkles (particularly large
wrinkles), or as an active ingredient in a pharmaceutical
composition to treat, improve or prevent conditions or symptoms
associated with heparanase activity.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the difference in heparanase activity in
normal human keratinocytes, under ultraviolet-irradiated and
non-irradiated conditions;
FIG. 2 is a set of immunostaining images for heparanase and heparan
sulfate in ultraviolet-irradiated and non-irradiated sections of
normal human buttock tissue;
FIGS. 3(a) and 3(b) are both schematic diagrams of pseudo-skin
models, where FIG. 3(a) shows a "heparan sulfate-decomposed model"
(heparan sulfate (-)) with no heparan sulfate present in the basal
membrane sheet, while FIG. 3(b) shows a "normal model" (heparan
sulfate (+)) with heparan sulfate present in the basal membrane
sheet;
FIG. 4 is a graph showing evaluation results for VEGF permeability
using the pseudo-skin models of FIGS. 3(a) and 3(b);
FIG. 5 is a set of photographs showing evaluation results for
angiogenesis using the pseudo-skin models of FIGS. 3(a) and 3(b);
and
FIG. 6 is a graph showing analysis results for blood vessel area in
the photograph of FIG. 5.
BEST MODE FOR CARRYING OUT THE INVENTION
The present inventors have searched for substances that can
efficiently inhibit heparanase activity and have screened for
various compounds as indicators of heparanase activity inhibition,
and as a result have completed this invention upon finding specific
resorcinol derivatives that significantly suppress heparanase
activity.
It has been completely unknown in the prior art that resorcinol
derivatives exhibit inhibiting action on heparanase activity.
JP-H2-49715A and JP2006-124358A disclose the use of specific
resorcinol derivatives as skin whiteners, and JP2007-254412A
discloses the use of specific resorcinol derivatives in external
preparations for skin for prevention or improvement of wrinkles,
but these publications contain no disclosure regarding the
inhibiting effect on heparanase activity.
In particular, JP2007-254412A teaches that the presence of heparin
(a type of heparan sulfate) in cells promotes disintegration of the
fascicular structure of collagen and leads to wrinkle formation
(see paragraph [0006]), and suggests that resorcinol derivatives
reduce intracellular heparin thus preventing disintegration of the
fascicular structure of collagen, but this is completely different
from the inhibiting action on heparanase activity by the resorcinol
derivatives disclosed in the present invention. That is, inhibition
of heparanase activity according to the invention causes
suppression of the decomposition of heparan sulfate chains of
heparan sulfate proteoglycan, so that the abundance of heparan
sulfate (heparin, etc.) in cells can be kept at a high level.
Specifically, a heparanase activity inhibitor of the invention
contains a 4-alkylresorcinol represented by formula (I) as an
active ingredient.
##STR00003##
In the formula, R represents a C1-6 linear or branched alkyl group.
Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl and
sec-hexyl. Preferred among these are C2-4 linear or branched alkyl
groups such as ethyl, n-propyl, isopropyl, n-butyl and isobutyl,
and especially isobutyl. That is, the 4-alkylresorcinol of formula
(I) is most preferably 4-isobutylresorcinol.
The method for producing the 4-alkylresorcinol of formula (I) is
not particularly restricted, but there may be mentioned a method of
Friedel-Crafts reaction between a saturated carboxylic acid or
saturated carboxylic acid halide and resorcinol in the presence of
a Lewis acid such as zinc chloride or aluminum chloride, and
reduction of the resulting 4-acylresorcinol with zinc
amalgam/hydrochloric acid (JP-H06-51619A and U.S. Pat. No.
2,093,778B), a method of using alumina as a catalyst for reaction
between resorcinol and n-hexanol in a liquid phase at
200-400.degree. C. to directly produce 4-n-hexylresorcinol
(GB1581428B), and a method of using one or more compounds selected
from among specific metal oxides and hydroxides as catalysts for
reaction of alcohol with resorcinol in a supercritical state to
produce 4-alkylresorcinol (JP2002-167344A).
The heparanase activity inhibitor of the invention may contain a
single 4-alkylresorcinol of formula (I) alone, but it may instead
contain 2 or more 4-alkylresorcinols of formula (I) in any desired
combination and proportion.
The content of the 4-alkylresorcinol of formula (I) in the
heparanase activity inhibitor of the invention is not particularly
restricted so long as it is an amount sufficient to effectively
exhibit inhibition against heparanase activity, and it may be
appropriately selected according to the purpose of the heparanase
activity inhibitor. Generally, however, the proportion of the
4-alkylresorcinol of formula (I) with respect to the entire
heparanase activity inhibitor is preferred to be usually at least
0.0001 mass % and especially at least 0.0001 mass %, and usually no
greater than 1 mass % and especially no greater than 0.2 mass %.
When two or more 4-alkylresorcinols of formula (I) are used, their
total amount must satisfy the aforementioned range.
The heparanase activity inhibitor of the invention may also contain
other desired components in addition to the 4-alkylresorcinol of
formula (I), so long as they do not substantially impair the
inhibiting effect of the 4-alkylresorcinol of formula (I) on
heparanase activity. Other components include other compounds with
inhibiting action on heparanase activity (other active components),
or medically acceptable carriers and/or adjuvants. Such other
components may be used alone, or 2 or more may be used in any
desired combination and ratio.
The heparanase activity inhibitor of the invention may be used as a
cosmetic, quasi drug, pharmaceutical composition or the like, or as
a compounding ingredient therein, although this is not
restrictive.
A pharmaceutical composition containing a heparanase activity
inhibitor of the invention as an active ingredient (a
pharmaceutical composition of the invention) can be used for
treatment, improvement or prevention of a condition or symptom
associated with heparanase activity. Here, the "condition or
symptom associated with heparanase activity" may be, for example,
skin aging, cancer cell proliferation or metastasis, angiogenesis,
or the like. Thus, the pharmaceutical composition of the invention
may be suitably used, for example, for improvement or prevention of
skin aging (anti-aging), suppression of cancer cell proliferation
or metastasis, or suppression of angiogenesis.
Natural aging is a major cause of skin aging from a macroscopic
viewpoint, but other causes such as oxidation, dryness and sunlight
(ultraviolet rays) are also direct factors related to skin aging.
The specific phenomenon of skin aging is known to be associated
with cellular damage due to reduction in mucopolysaccharides
including hyaluronic acid, collagen crosslinking reaction and
ultraviolet rays.
A great deal of research is being carried out with the aim of
inhibiting or improving skin wrinkles, fine wrinkles, sagging and
the like, caused by skin damage or skin aging due to ultraviolet
exposure. As a result, efficacy has been demonstrated for promoting
hyaluronic acid production (JP2001-163794A), suppressing production
and activation of matrix metalloproteinases (MMP) (JP2000-503660X),
promoting production of collagen and inhibiting esterase activation
(JP-H11-335235A), suppressing angiogenesis (WO03/84302 and Japanese
Patent Application No. 2003-581562), and suppressing
lymphangiectasis (K. Kajiya et al., Am. J. Pathol., 2006, 169(4):
1496-1503).
Such research is largely divided into efforts to prevent and
improve fine wrinkles, with focus on the epidermis or epidermal
cells, and efforts to prevent and improve large wrinkles, with
focus on suppressing changes in the dermis including blood vessels
or lymphatic vessels. Propagation of changes in the epidermis to
the dermis leads to alteration of the dermis, including the blood
vessels and lymphatic vessels, and heparanase is intricately
involved in the process.
The present inventors have in fact previously demonstrated that a
significant anti-wrinkle effect is obtained by coating a fine
wrinkle model with a heparanase activity inhibitor
(PCT/JP2009/056717).
As explained in detail in the examples, the present inventors have
demonstrated that irradiation of cultured normal keratinocytes with
ultraviolet rays results in activation of the heparanase of the
normal keratinocytes (see FIG. 1). It was also demonstrated that
irradiation of human skin with ultraviolet rays increases the
amount of heparanase in the epidermis, and reduces heparan sulfate
in the basal membrane (see FIG. 2). It was thereby shown that
heparanase activation occurs not only in fine wrinkle models but
also by ultraviolet rays.
In addition, since basal membrane heparan sulfate is decomposed
upon activation of heparanase, the present inventors prepared, as
pseudo-skin models, a normal model containing heparan sulfate in a
basal membrane and a heparan sulfate-decomposed model containing no
heparan sulfate in the basal membrane, and evaluated VEGF
permeability and angiogenesis. As a result, it was shown that VEGF
permeability was increased and angiogenesis was induced in the
heparan sulfate-decomposed model, compared to the normal model (see
FIGS. 3 to 6).
Yano et al. have previously indicated that ultraviolet ray-induced
induction of angiogenesis in the dermis and alteration of the
dermis are important for formation of large wrinkles (Japanese
Patent Application No. 2002-580892), and have found that heparanase
is an enzyme intricately involved in not only fine wrinkles but
also large wrinkles. That is, inhibition of heparanase activity is
effective for not only preventing fine wrinkles due to dryness but
also large wrinkles due to prolonged sun exposure.
The term "anti-aging", as used herein, means preventing and
improving wrinkles, sagging and hardening of skin by suppressing
alteration of skin caused by release of heparan sulfate-binding
growth factors due to decomposition of proteoglycan heparan sulfate
in the basal membrane by aging or photoaging, and specifically
suppressing epidermal differentiation abnormalities, dermis
angiogenesis, lymphangiectasis and elastin breakdown, to maintain
an elastic, youthful and healthy state of skin.
The route of administration and dosage form of the pharmaceutical
composition of the invention are not restricted, and may be
selected as appropriate for the purpose. Examples of routes of
administration include oral administration, parenteral
administration (such as intravenous administration and
intraperitoneal administration), local administration (such as skin
application) and the like. For oral administration, the dosage form
may be a solid preparation such as a tablet, coated tablet,
sugar-coated tablet, granules, powder, capsule (for example, a hard
or soft gelatin capsule), or a liquid preparation (solution or
suspension) such as an internal liquid drug or syrup. For a
parenteral administration, it may be in the form of an injection or
the like. For local administration, it may be a form in which a
solution system, solubilized system, emulsified system,
powder-dispersed system, water/oil two-layer system,
water/oil/powder three-layer system or the like, is prepared as a
patch, ointment, cream, latex, cosmetic water, gel or aerosol.
The content of the heparanase activity inhibitor of the invention
in a pharmaceutical composition of the invention is also not
restricted, and may be appropriately selected according to the
purpose, dosage form and route of administration of the
pharmaceutical composition. When the pharmaceutical composition of
the invention is an external preparation for skin, for example, the
content of the heparanase activity inhibitor of the invention is
preferred to be usually in the range of at least 0.0001 mass %, and
especially at least 0.0001 mass %, and usually no greater than 1
mass % and especially no greater than 0.2 mass %, as the dry mass
(solid mass) of the total external preparation for skin. If the
content is less than this range, the effect of the heparanase
activity inhibitor of the invention will tend to be insufficient,
and if it exceeds this range, no further effect may be expected
commensurate with the increased content, and formulation will also
tend to become difficult.
The pharmaceutical composition of the invention may also contain
one or more other desired components in addition to the heparanase
activity inhibitor of the invention, so long as the inhibiting
effect on heparanase activity by the heparanase activity inhibitor
of the invention is not substantially impaired. There are no
particular restrictions on such other components, and they may be
appropriately selected according to the purpose, dosage form and
route of administration of the pharmaceutical composition, but
medically acceptable carriers and/or adjuvants may be mentioned as
examples. Examples of adjuvants include diluents, binders,
disintegrators, thickeners, dispersing agents, reabsorption
accelerators, taste correctives, buffering agents, surfactants,
dissolving aids, preservatives, emulsifiers, isotonizing agents,
stabilizers and pH regulators.
As specific examples, when the pharmaceutical composition of the
invention is to be used as an external preparation for skin,
components commonly used in external preparations, such as skin
whiteners, humectants, antioxidants, oil components, ultraviolet
absorbers, surfactants, thickeners, alcohols, powder constituents,
coloring agents, aqueous components, water or various skin nutrient
preparations, may be appropriately added as necessary. In addition,
there may also be added appropriate amounts of metal ion chelators
such as disodium edetate, trisodium edetate, sodium citrate, sodium
polyphosphate, sodium metaphosphate or gluconic acid, antiseptic
agents such as methylparaben, ethylparaben or butylparaben,
caffeine, tannin, verapamil, tranexamic acid or their derivatives,
licorice extract, drug agents such as glabridin, Chinese quince
fruit hot water extract, galenicals, tocopherol acetate,
glycyrrhizic acid and its derivatives or salts, skin whiteners such
as vitamin C, magnesium ascorbate phosphate, glucoside ascorbate,
arbutin or kojic acid, saccharides such as glucose, fructose,
mannose, sucrose or trehalose, and vitamin A derivatives such as
retinoic acid, retinol, retinol acetate or retinol palmitate.
On the other hand, when the heparanase activity inhibitor of the
invention is to be used in a cosmetic or quasi drug, it is
preferably used as an active ingredient of a wrinkle improving
agent. A wrinkle improving agent containing the heparanase activity
inhibitor of the invention as an active ingredient (a wrinkle
improving agent of the invention) may be used to prevent or
suppress formation of wrinkles. As mentioned above, wrinkles are
largely classified as fine wrinkles formed in the epidermis by
dryness and the like, and large wrinkles formed in the dermis by
ultraviolet rays and the like, and a wrinkle improving agent of the
invention may be applied to either type and is particularly
effective for improving large wrinkles caused by ultraviolet
rays.
The route of administration and dosage form of the wrinkle
improving agent of the invention are not restricted, and may be
selected as appropriate for the purpose. Oral administration and
local administration may be mentioned as examples of routes of
administration. Examples of dosage forms include the various dosage
forms mentioned above, for a pharmaceutical composition, and
addition to foods or beverages, for oral administration.
The content of the heparanase activity inhibitor of the invention
in a wrinkle improving agent of the invention is also not
restricted, and may be appropriately selected according to the
purpose, dosage form and route of administration of the wrinkle
improving agent.
The wrinkle improving agent of the invention may also contain one
or more other desired components in addition to the heparanase
activity inhibitor of the invention, so long as the inhibiting
effect on heparanase activity by the heparanase activity inhibitor
of the invention is not substantially impaired. There are no
particular restrictions on other components, and they may be
appropriately selected according to the purpose of use, the dosage
form and the route of administration of the wrinkle improving
agent.
The present invention has been explained with concrete examples,
with the understanding that these are merely for illustration and
that the invention may incorporate any desired modifications that
fall within the scope of the claims of the invention.
The present invention will now be explained in greater detail with
reference to examples, with the understanding that the invention is
not meant to be limited to these examples.
EXAMPLES
Example 1
Evaluation Based on Heparanase Activity Inhibition Rate
A431 cells (human epithelial carcinoma cells) were cultured in 10%
serum-containing DMEM (Dulbecco's modified Eagle medium). The
cultured cells were solubilized in lysis buffer (50 mM Tris, 0.5%
TritonX-100, 0.15 M sodium chloride, pH 4.5) and collected with a
scraper, and then pipetted and allowed to stand on ice for 30
minutes. This was followed by centrifugation at 10,000 rpm for 10
minutes to remove the insoluble portion, and the supernatant was
recovered as cell extract. The amount of protein in the cell
extract was measured with a BCA protein assay kit (BCA Protein
Assay Kit, PIERCE, Calif. 46141).
The A431 cell extract was then diluted to 500 .mu.g/mL with assay
buffer (50 mM HEPES, 50 mM sodium acetate, 150 mM sodium chloride,
9 mM calcium chloride, 0.1% BSA). Next, the test compound was
dissolved in DMSO and added to the diluted cell extract in
proportions of 0.0005 mass %, 0.005 mass % and 0.05 mass %, and
these were mixed to prepare sample solutions (DMSO final
concentration: 5%). A control solution was prepared by mixing DMSO
with the diluted cell extract to a final concentration of 5%. The
sample solution and control solution were each seeded in a
biotinylated heparan sulfate-immobilized plate at 100 .mu.L/well.
After reaction at 37.degree. C. for 2 hours and rinsing 3 times
with PBS-T, 10,000-fold diluted HRP-avidin (Vector, A-2004)/PBS-T
was added at 100 .mu.L/well, and reaction was continued at
37.degree. C. for 1 hour. After further rinsing 3 times with PBS-T,
TMB reagent (BIO-RAD, 172-1066) was added at 100 .mu.L/well and
reacted therewith, the reaction was terminated with 1N sulfuric
acid, and the absorbance at 475 nm (OD475) was measured.
Also, DMSO was added to a serial diluent prepared with the
aforementioned A431 cell extract assay buffer (cell extract
concentrations: 500 .mu.g/mL, 50 .mu.g/mL, 5 .mu.g/mL, 0.5
.mu.g/mL), to a final concentration of 5% without addition of the
test compound, to obtain a mixture (solution for calibration
curve). The solution for the calibration curve was subjected to
treatment by the same procedure described above, from seeding of
the biotinylated heparan sulfate-immobilized plate, and the OD475
was measured.
Next, a calibration curve for protein concentration was drawn based
on the OD475 value of the solution for the calibration curve, and
this calibration curve was used to calculate the protein
concentration of each sample solution from the OD475 value of a
sample solution obtained by adding the test compound at different
addition concentrations. The protein concentration was calculated
in the same manner for the control solution. The heparanase
activity inhibition rate of each sample solution was determined
from the ratio of the protein concentration of each sample solution
and the protein concentration of the control solution (%).
The details regarding this procedure are described in
JP2003-502054X.
The heparanase activity inhibiting effect of 4-isobutylresorcinol
was tested by the procedure described above. The results are shown
in Table 1. Table 1 shows that 4-isobutylresorcinol exhibits an
inhibition rate of 37.64% even at an addition concentration of
0.0005%, and 94.74% at an addition concentration of 0.05%, and thus
effectively inhibits heparanase activity.
TABLE-US-00001 TABLE 1 Heparanase activity inhibition rates
Compound Added concentration Inhibition rate 4-Isobutylresorcinol
0.0005% 37.64% 4-Isobutylresorcinol 0.005% 75.98%
4-Isobutylresorcinol 0.05% 94.74%
Example 2
Evaluation of Change in Heparanase Activity by Ultraviolet
Radiation
Normal human keratinocytes were cultured with EpiLife normal
keratinocyte medium. The culture medium was temporarily stationed
in PBS and then irradiated with 50 mJ UVB, and after culturing for
1 hour, 2 hours and 4 hours, the cells were solubilized with lysis
buffer and used as sample solutions in the ultraviolet irradiation
group. Also, medium was temporarily stationed in PBS without
ultraviolet irradiation, for use as a control solution. The sample
solutions and control solution were used for treatment in the same
manner as Example 1, and the OD475 was measured. The heparanase
activities were evaluated in the same manner as Example 1, based on
the obtained OD475 values. The results are shown in FIG. 1. It was
shown that heparanase was significantly activated in the
ultraviolet irradiation group compared to the non-irradiated
control.
Immunostaining of Heparanase and Heparan Sulfate in
Ultraviolet-Irradiated Human Skin
Human buttock skin (20-year-old) was irradiated with 2MED
ultraviolet rays, and after 2 days the irradiated section and
surrounding non-irradiated buttock skin were biopsied, and a
paraffin block was formed by the AMeX method. A 3 .mu.m tissue
section was formed, and the heparanase and heparan sulfate were
immunostained. The obtained immunostaining image is shown in FIG.
2. The amount of heparanase was clearly increased and the heparan
sulfate content was reduced in the ultraviolet irradiated section,
compared to the non-irradiated section.
Evaluation of VEGF Permeability and Angiogenesis with and without
Heparan Sulfate
After heating and dissolving 2 mg of heparan sulfate and 10 mg of
agarose in 1 ml of PBS (1% agarose solution), it was coated with an
insert (24-well Transwell by Corning, Inc.) to form a heparan
sulfate-containing sheet. As a control, a sheet containing no
heparan sulfate was formed by the same procedure, except that
agarose alone was used, without using heparan sulfate. The insert
interior was selected for the epidermis side, the sheet as the
basal membrane, and the well on the dermis side, to prepare a
pseudo-skin model (FIG. 3a,b).
The obtained pseudo-skin model can be used as an evaluation system
for evaluating VEGF permeability and angiogenesis, based on the
presence or absence of heparan sulfate in the sheet selected as the
basal membrane (hereinafter referred to as "basal membrane sheet").
In the explanation which follows, the pseudo-skin model containing
heparan sulfate in the basal membrane sheet is referred to as the
"normal model", and the pseudo-skin model containing no heparan
sulfate in the basal membrane sheet is referred to as the "heparan
sulfate-decomposed model".
First, for evaluation of the VEGF permeability, a 10 .mu.g/mL VEGF
aqueous solution was added to the epidermis side (insert interior)
of each model and allowed to stand for 3 hours at room temperature,
and the VEGF concentration in the well on the dermis was detected
with a VEGF ELISA kit (R&D systems). The results are shown in
FIG. 4. The VEGF permeation was significantly reduced in the normal
model compared to the heparan sulfate-decomposed model.
Next, for evaluation of angiogenesis, a 100 .mu.g/mL VEGF aqueous
solution was added to the epidermis side (insert interior) of each
model, and set in an angiogenesis kit (Kurabo Industries, Ltd.) for
culturing for 11 days, after which an optical microscope photograph
of the culture was taken. The obtained image is shown in FIG. 5.
Notable angiogenesis was observed in the heparan sulfate-decomposed
model in a concentration dependent manner, while no angiogenesis
was observed in the normal model.
Angiogenesis kit analysis software (Kurabo Industries, Ltd.) was
used to analyze the blood vessel area in the image of FIG. 5. The
results are shown in FIG. 6. A notable increase in blood vessel
area was observed in the heparan sulfate-decomposed model compared
to the normal model, demonstrating significant angiogenesis.
INDUSTRIAL APPLICABILITY
The present invention can be suitably used in fields of
pharmaceutical compositions and the like, for the purpose of
treatment, improvement or prevention of conditions or symptoms
associated with heparanase activity, and specifically for
improvement or prevention of skin aging (anti-aging), for wound
healing, for suppression of proliferation or metastasis of cancer
cells, and for suppression of angiogenesis.
* * * * *